Effect of hyaluronidase and PEG chain conjugation on the biologic and antitumor activity of RNase A

Abstract

Subcutaneous application of bovine RNase A conjugated to HYase (bovine hyaluronidase), polyethylene glycol (PEG) and HYase+PEG resulted in a marked reduction of the width of the spermatogenic layers of the mouse testes. The number of sperms in caput epididymidis was significantly decreased in mice injected with conjugated RNase A. There was not any significant embryotoxic effect of free RNase A even conjugated with HYse, PEG and HYse+PEG. The immunogenicity, expressed in production of antibodies against free RNase A or conjugates with PEG, was very low. However, the immunogenic action of this enzyme conjugated only to HYase was much higher and produced the same immunogenicity as HYase itself. The immunogenic effect of RNase A+HYase conjugate decreased when PEG was joined to this conjugate. The inhibitory effect of RNase A conjugated to HYase, PEG and HYase+PEG on human ML-2 cells studied in vitro, was practically ineffective. On the other side, when RNase A conjugated to HYase or PEG was administered intraperitoneally into the mice bearing human melanoma, the antitumor effect was pronounced.

Introduction

Various biological aspects of bovine pancreatic ribonuclease (RNase A) has been intensively studied for many years [7], [29]. Some antitumor effect of this enzyme was found shortly after its isolation [19]. However, the inhibition of its antitumor and other biological activities by ribonuclease inhibitor (RI) in cytosol was the main reason for the low cytotoxic activity of this RNase. From this reason, an attention of scientists in the last years was focussed on the mutation of recombinant RNase A, human pancreatic RNase (HPRNase), and on efforts to block the binding of RI to this molecule. Beside producing a simple mutation in the synthetic gene of RNase A [20] the conjugation of this monomeric ribonuclease with transferrin, poly [N-(2 hydroxypropyl) methacrylamide] or polyethylen glycol (PEG) seems to help to solve this relevant problem [25], [32], [36], [38].

The effects of bovine even ovine testicle hyaluronidase (HYase), a matrix-degrading enzyme, were studied in the last years in extracts from various primary tumors and regional metastases [2]. They were also used as additives to induction chemotherapy and enhancement of the permeation of chemotherapeutics into tumor tissue with a matrix rich on hayluronic acid [1], [17], [18], [28] or hyaluronan synthase [12]. The further positive effect of HYase to tumors has been directed to use this enzyme as chemosensitizer, which acts as antiadhesive agent rendering solid resistant tumors more susceptible to conventional therapeutic drugs [34]. Notably, hyaluronidase increases tumor necrosis factor (TNF) sensitivity in several types of cancer cells, counteracts transforming growth factor beta (TGF-beta), mediated TNF-resistance and suppresses TGF-beta 1 gene expression in L 929 cells which are enable to resist TNF killing [5]. The possibility to modulate the expression of CD 44 (transmembrane glycoproteins) expressed on a wide variety of cells, including cancer cells, by HYase seems to be also positive for the prevention of tumor growth [13], [30], [35], [40], [30], [13].

It is also demonstrated that the membrane-associated HYase can act in vivo, and that hyaluronan, which is synthesized by the tumor stroma, can be made soluble and reduced to a smaller size by tumor cells before being internalized and further digested [8]. HYase is well tolerated by the test animals [33]. In a prospective pilot study, patients with advanced inoperable squamous cell carcinoma of the head and neck were treated with polychemotherapy and hyaluronidase combined with radiation therapy. The published results suggested that combined therapy with vindesine, cisplatin, HYase, and radiation were highly effective against this squamous cell cancer [17].

Even though some negative experiments showed that elevated HYase levels correlate with tumor metastasis and angiogenesis [9], [21], [22], [23], [39] we conjugated bovine HYase to RNase A. Beside this conjugate we joined to the RNase+HYase complex the PEG chain polymer yet. These two RNase polymerized conjugates were studied biologically.